Allele-specific silencing therapy for Dynamin 2-related diseases

ABSTRACT

The invention relates to an allele specific siRNA able to silence the expression of only one allele of a heterozygous DNM2 gene, for treating diseases caused by heterozygous mutation and/or overexpression of Dynamin 2.

FIELD OF THE INVENTION

The present invention relates to an allele specific siRNA able tosilence the expression of only one allele of a heterozygous DNM2 gene,for treating diseases caused by heterozygous mutation and/oroverexpression of Dynamin 2.

BACKGROUND

Dynamin 2 is a ubiquitously expressed protein that belongs to thesuperfamily of large GTPases. Dynamin 2 (DNM2) acts as a mechanochemicalscaffolding molecule that deforms biological membranes leading to therelease of nascent vesicles. At the plasma membrane DNM2 is involved inclathrin-dependent and clathrin-independent endocytosis. This protein isalso involved in the formation of vesicles from endosomes andtrans-Golgi network. Several studies have highlighted the role ofDynamin 2 as regulator of actin and microtubule cytoskeletons.

Several dominant genetic diseases are caused by heterozygous mutationsof the DNM2 gene coding for said Dynamin 2 protein. In particular,autosomal dominant centronuclear myopathy (AD-CNM) results frommutations in the DNM2 gene. AD-CNM is a rare congenital myopathycharacterized by the high incidence of centrally placed nuclei in musclefibers in absence of regenerative process. The AD-CNM is associated witha wide clinical spectrum from severe-neonatal to mild-adult forms. Ingeneral, motor milestones are delayed and diffuse skeletal muscleweakness mainly involves facial and limb muscles. Muscle weakness isslowly progressive but loss of independent ambulation may occur duringthe fifth decade. In the severe and early-onset CNM, paediatric patientsusually have generalized weakness, hypotonia, moderate degree of facialweakness with open mouth, ptosis and ophthalmoplegia. No curativetreatment is available for the AD-CNM.

Mutations in the DNM2 gene are also involved in rare cases ofCharcot-Marie-Tooth disease and Hereditary Spastic Paraplegia.Charcot-Marie-Tooth disease (CMT) is a hereditary motor and sensoryneuropathy characterized by progressive loss of muscle tissue and touchsensation across various parts of the body. Hereditary spasticparaplegia (HSP) is an inherited disease characterized by lowerextremity spasticity and weakness occurring in variable proportion.

About thirty heterozygous mutations in the DNM2 gene were identified asinvolved in AD-CNM, CMT, or HSP diseases, for which there is noavailable curative treatment.

Overexpression of DNM2 in absence of mutation is also involved in somepathophysiological mechanisms of other diseases such X-linked myotubularmyopathy or cancers, for example prostate cancer and pancreatic cancer.

Therefore, there remains an urgent need for therapeutic compounds andmethods for the treatment of diseases caused by heterozygous mutationsand/or overexpression of DNM2.

Some research teams have developed strategies to inhibit Dynamin 2 usingconventional siRNA or pharmacological inhibitors, in order to treat DNM2overexpression-linked pathologies. The present invention relates to animprovement of these strategies which is able to inhibit mutated oroverexpressed Dynamin 2, while preserving a sufficient amount offunctional DNM2 allowing a normal cellular function.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides an allele specific siRNA(AS-siRNA) able to silence the expression of only one allele of aheterozygous DNM2 gene in a cell. In a particular embodiment, theAS-siRNA of the invention is of 19-23 base pairs in length, preferably19.

In a particular embodiment, the AS-siRNA of the invention is able tosilence the expression of only one allele of a heterozygous DNM2 gene ina cell, the DNM2 gene being heterozygous for the presence of anon-pathological polymorphism, and/or being heterozygous for thepresence of a disease-causing mutation.

In particular, the AS-siRNA of the invention targets a region of a DNM2gene transcript comprising said non-pathological polymorphism. In aparticular embodiment, said non-pathological polymorphism is on the sameallele as the disease-causing mutation. More specifically, saidnon-pathological polymorphism is rs2229920 (C or T) or rs12461992 (A orT), preferably rs2229920 (C or T).

In a more particular embodiment, the AS-siRNA of the invention is of 19base pairs in length and comprises a mismatch. This mismatch enables theAS-siRNA to specifically target and silence only the allele carryingsaid polymorphism, while preserving the other allele. In a particularembodiment, the position of the mismatch is located at position N16 orN17 from 5′ end of the sense strand of said AS-siRNA, preferably atposition N17.

In particular, the AS-siRNA comprises a sense strand selected in thegroup consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ IDNO:4, preferably SEQ ID NO:1 or SEQ ID NO:2.

In another particular embodiment, the AS-siRNA of the invention targetsa region of a DNM2 gene transcript comprising said disease-causingmutation. More specifically, the disease-causing mutation is 1393C>T;c.1105C>T, c.1106G>A, c.1393C>T, c.1856C>T or c.1948G>A, preferably1393C>T. In a particular embodiment, the AS-siRNA of the invention is of19 base pairs in length and comprises a mismatch. This mismatch enablesthe AS-siRNA to specifically target and silence the mutant allele, whilepreserving the wild type allele. In a particular embodiment, theposition of the mismatch is located at position N9, N10, N11, N12, N15or N16 from 5′ end of the sense strand of said AS-siRNA, preferably atposition N9 or N10 and more preferably at position N9.

In particular, the AS-siRNA comprises a sense strand selected in thegroup consisting of: SEQ ID NO:5 to SEQ ID NO:10, preferably SEQ ID NO:5or SEQ ID NO:6, and more preferably SEQ ID NO:5.

In some embodiments, the AS-siRNA of the invention is able to reduceexpression of DNM2 mRNA and/or DNM2 protein by 20-60%, preferably around50%.

Another aspect of the invention relates to a vector encoding theAS-siRNA of the invention, the vector being preferably a plasmid or aviral vector, such as an AAV vector.

The invention also relates to a target cell, which is transfected ortransduced with the vector of the invention.

In a further aspect, the invention provides an in vitro method forsilencing the expression of the mutated allele of DNM2 gene withoutsilencing the expression of the wild type allele of the DNM2 gene in atarget cell, comprising introducing in said target cell an AS-siRNA or avector of the invention.

According to another aspect, herein is disclosed an AS-siRNA, a vectoror a cell of the invention for use in a method for treating a diseaseassociated with an overexpression of Dynamin 2, preferably for treatingX-linked myotubular myopathy, or cancer such as prostate cancer andpancreatic cancer.

Another aspect of the invention relates to an AS-siRNA, a vector or acell of the invention for use in a method for treating a disease inducedby a disease-causing mutation(s) in the DNM2 gene, preferably fortreating autosomal dominant centronuclear myopathy, T-cell acutelymphoblastic leukemia, Charcot-Marie-Tooth disease or HereditarySpastic Paraplegia, and more preferably for treating autosomal dominantcentronuclear myopathy.

Other aspects and embodiments of the invention will be apparent in thefollowing detailed description.

LEGENDS OF THE FIGURES

FIG. 1. Screening for allele-specific siRNA in heterozygous MouseEmbryonic Fibroblasts (MEF). (A) Wild-type (WT; SEQ ID NO: 11) andmutated (R465W; SEQ ID NO: 12) mRNA sequences in the region of thenucleotide change are indicated as well as the sequences of the 19possible siRNA targeting the single point mutation (siRNA sense strandwhich is not complementary to the mRNA sequence). Arrows show the 12siRNA assessed in this study. (B) EcoNI digestion profile on agarose gelelectrophoresis of the Dnm2 RT-PCR product centred on the mutatednucleotide. The siRNA are numbered relative to the position of themismatch introduced between the siRNA and the WT sequences. Sc: ScramblesiRNA. NT: non transfected cells. Histogram represents mean.+−.SEM ofcalculated HTZ/WT ratio for each siRNA. * P<0.05 using a Mann-WhitneyU-test compared to Scramble value (n=5).

FIG. 2. Allele-specific si9 and si10 in heterozygous Mouse EmbryonicFibroblasts (MEF). (A) Agarose gel of RT-PCR products for Dnm2 and GapdhmRNA and quantification of Dnm2 expression normalized to Gapdh used as aloading control. (B) Sequence of Dnm2 amplicons from cells transfectedwith si9, si10 and Scramble siRNA. Squares indicate the mutatednucleotide (N=T and A with Scramble siRNA) (C) EcoNI digestion profileof the Dnm2 amplicon on agarose gel electrophoresis and quantificationof the Mutant/WT ratio. (D) Representative western blot against Dnm2 intreated and control cells and quantification of signal by densitometry.Gapdh was used as loading control. Sc: Scramble siRNA. NT: nontransfected cells. In A, C, and D, histograms represent mean±SEM. **P<0.001 using a Mann-Whitney U-test compared to Scramble values (n=5).

FIG. 3. si9 and si10 are allele-specific siRNAs in Mouse Myoblast. Cellswere transfected with siRNAs at 100 nM for 48 h. (A) Representativeimages of Desmin immunostaining in immortalized mouse myoblast. Scalebars=50 μm. (B) Dnm2 and Gapdh semi-quantitative RT-PCR products fromsiRNA transfected myoblasts and quantification of Dnm2 expressionnormalized to Gapdh. (C) Quantification of the mutated and WT Dnm2transcripts after RT-PCR and EcoNI digestion and normalization relativeto Gapdh expression. Scatter plots bars represent mean±SEM. * P<0.05using a one-tailed Mann-Whitney U-test compared to scramble values(n=4).

FIG. 4. Contractile properties in TA muscles. P0: absolute force. sP0:specific maximum tetanic force. mo: month. n: number of analyzedmuscles. Statistical comparison was performed using a Mann-WhitneyU-test. Statistical analysis vs WT: a: P<0.05 and b: P<0.001.Statistical analysis vs HTZ noSh: c: P<0.05, d: P<0.01 and e: P<0.001.

FIG. 5. Molecular and histological characterization of shAAV-transducedmuscles in mice. (A) Agarose gel of RT-PCR products for Dnm2 and GapdhmRNA from shAAV-transduced HTZ TA muscles and quantification of Dnm2expression normalized to Gapdh used as a loading control. WT muscleswere included as control. Histograms represent mean±SEM. * P<0.05 usinga Mann-Whitney U-test compared to nosh value (n=5) (B) Sequence of Dnm2amplicons from TA muscles transduced with sh9-, sh10- and nosh-AAV.Squares indicate position of the mutated nucleotide (N=T and A) (C)EcoNI digestion profile of the Dnm2 amplicon on agarose gelelectrophoresis and quantification of the mutant/WT ratio. Histogramsrepresent mean±SEM. * P<0.05 using a Mann-Whitney U-test compared tonosh value (n=5). (D) Representative histochemical staining of TAsections from WT and HTZ mice transduced with shAAV or empty AAV. HE:hematoxilin eosin staining. DPNH: Reduced diphosphopyridine nucleotidediaphorase staining. Scale bars=50 μm. (E) Quantification of thefrequency of fibre size in transduced HTZ TA muscles. WT muscles wereused as control (n=3 per condition).

FIG. 6. Allele-specificity of si9 and si10 in patient-derivedfibroblasts. (A) Agarose gel of RT-PCR products for Dnm2 and Gapdh mRNAand quantification of Dnm2 expression normalized to Gapdh used as aloading control. (B) Sequence of Dnm2 amplicons from cells transfectedwith si9, si10 and Scramble siRNA. Squares indicate the mutatednucleotide (N=T and C with Scramble and si10 siRNA) (C) PfoI digestionprofile of the Dnm2 amplicon on agarose gel electrophoresis andquantification of the Mutant/WT ratio. (D) Representative western blotagainst Dnm2 in treated and control cells and quantification of signalby densitometry. Gapdh was used as loading control. Sc: Scramble siRNA.NT: non transfected cells. In A, C, and D, histograms representmean±SEM. * P<0.05 and ** P<0.001 using a Mann-Whitney U-test comparedto Scramble values (n=5).

FIG. 7. Allele-specific silencing induced by si9 in human fibroblasts at100 nM. (A) Agarose gel electrophoresis of Dnm2 and HPRT RT-PCR products48 hours after transfection of siRNA at 100 nM. NT: non-transfectedcells. Histogram represents mean±SEM of DNM2 normalized to HPRT. **P<0.001 using a Mann-Whitney U-test compared to scramble values (n=5).(B) BglI digestion profile of the DNM2 amplicon on agarose gelelectrophoresis and quantification of the Mutant/WT ratio. Histogramrepresents mean±SEM. ** P<0.001 using a Mann-Whitney U-test compared toscramble values (n=5). (C) Representative pictures of human fibroblasts48 h after transfection with si9 and scramble siRNA at 100 nM.

FIG. 8. Transferrin uptake assay in fibroblasts. (A) Transferrin uptakeassay in patient-derived fibroblasts. Transferrin uptake was quantifiedafter 15 min of incubation at 37° C. Histogram represents mean±SEM (n=65cells from 2 independent experiments for each cell line) and statisticalanalysis was performed using a Student-t test (*** p<0.0001 versus the 2control cell lines). (B) Transferrin uptake assay in healthy controlfibroblasts. Transferrin uptake was quantified after 15 min ofincubation at 37° C. Histogram represents mean±SEM (n=65 cells from 2independent experiments for each cell line) and statistical analysis wasperformed using a Student-t test showing no significant differencebetween scramble and si9 values for each control cell line.

FIG. 9. Development of allele-specific siRNA against a non-pathogenicpolymorphism in human cells. (A) Agarose gel of RT-PCR products for DNM2and HPRT mRNA from human fibroblasts transfected by siRNA (30 nM for 48hours) directed against the two versions of the heterozygouspolymorphism (T or C). Scramble: Scramble siRNA. NT: non transfectedcells. (B) Quantification of the DNM2/HPRT ratio. Histogram representsmean±SEM. ** P<0.01 and *** P<0.0001 using a Mann-Whitney U-testcompared to Scramble values (n=8 for NT, Scramble, si17-713C. n=6 forsi16-713C. n=2 for si17-713T). Sc: Scramble siRNA.

FIG. 10. Allele-specificity of siRNA against the heterozygouspolymorphism. (A) Representative agarose gels of DNM2 RT-PCR productsfrom cells transfected by siRNA (30 nM for 48 hours) or innon-transfected cells (NT). In this patient-derived cell line, themutation is in frame with the version C of the polymorphism. The RT-PCRproducts are submitted to BglI digestion to discriminate between WTnon-digested allele and mutated digested allele. (B) Quantification ofthe mutant/WT and WT/HPRT ratios. Histograms represent mean±SEM. **P<0.01 and *** P<0.0001 using a Mann-Whitney U-test compared to Scramblevalues (n=8 for NT, Sc, si17-713C. n=6 for si16-713C. n=2 forsi17-713T). Sc: Scramble siRNA. NT: non-transfected cells.

FIG. 11. Modification of the length of the allele-specific siRNA againstthe heterozygous polymorphism. (A) Sequence of the siRNA (si-17C/21 isSEQ ID NO: 34, si-19C/21 is SEQ ID NO: 36. and si-20C/22 is SEQ ID NO:38). The underlined sequence shows the sequence of the 19-bp-lengthsiRNA with the polymorphism (C in bold case) at the position 17. In boldare indicated the additional bases in the modified siRNA. (B) Agarosegel of total DNM2 RT-PCR products (top panel), RT-PCR products digestedby BglI (middle panel) and HPRT RT-PCR product (bottom panel). (C)Quantification of the total DNM2/HPRT ratio. (D) Quantification of theallele C/allele T ratio. In C and D, Histograms represent mean.+−.SEM(n=2).

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based on the unexpected discovery of allelespecific siRNA (AS-siRNA) able to efficiently silence the expression ofonly one allele of a heterozygous DNM2 gene in a cell, said AS-siRNAbeing used for the treatment of DNM2-related diseases.

Dynamin 2 is encoded by the DNM2 gene (Gene ID 1785). More precisely,the DNM2 gene is located within the short arm of chromosome 19 atposition 13.2 (19p13.2). The dynamin 2 gene or gene products are alsoknown by other names, including but not limited to CMT2M, CMTDI1,CMTDIB, DI-CMTB, DYN2, DYN2 HUMAN, dynamin II, DYNII. DNM2 has animportant role in endocytosis and in the cell's structural framework(cytoskeleton). The protein interacts with multiple parts of thecytoskeleton, including microtubules and actin, which organize intofilaments to provide structure. These parts of the cytoskeleton areinvolved in movement of molecules within the cells, cell shape, cellmobility, and attachment of cells to one another or to extracellularmatrix. An alteration in the DNM2 gene may thus disrupt endocytosis andinterfere with the arrangement or dynamics of cytoskeletons leading toabnormal cellular function. As previously described, several dominantgenetic diseases are caused by heterozygous mutations of the DNM2 genesuch as autosomal dominant centronuclear myopathy, Charcot-Marie-Toothdisease and Hereditary Spastic Paraplegia. Overexpression of DNM2 isalso pathological and involved in some pathophysiological mechanisms ofother diseases such as X-linked myotubular myopathy or cancers, forexample prostate cancer and pancreatic cancer.

The present inventors have investigated a therapeutic approach based onthe specific suppression of the expression of only one allele of DNM2,preserving the other DNM2 allele. On one hand, this strategy aims toreduce in a controlled way the DNM2 expression level, in the case ofdiseases related to overexpression of DNM2. On the other hand, thisstrategy would be useful in autosomal dominant inherited diseases due toheterozygous mutation in the DNM2 gene, by specifically inhibiting theexpression of a mutant allele without reducing the level of the wildtype DNM2 allele which is required for a normal cellular function. Withthis objective, the present inventors discovered very efficient allelespecific siRNAs able to inhibit, in a controlled way, only one allele ofa heterozygous DNM2 gene in a cell.

A first aspect of the present invention is therefore an allele specificsiRNA (AS-siRNA) able to silence the expression of only one allele of aheterozygous DNM2 gene in a cell.

RNA interference is a biological process in which RNA molecules inhibitgene expression, typically by causing the destruction of specific mRNAmolecules. An interfering RNA is therefore an RNA which is capable ofdown-regulating the expression of the targeted protein. For example, itencompasses small interfering RNA (siRNA), double-stranded RNA (dsRNA),single-stranded RNA (ssRNA), and short hairpin RNA (shRNA) molecules.RNA interference designates a phenomenon by which dsRNA specificallysuppresses expression of a target gene at post-transcriptional level. Innormal conditions, RNA interference is initiated by double-stranded RNAmolecules (dsRNA) of several thousands of base pair length. In vivo, adsRNA introduced into a cell is cleaved by an enzyme called DICER into amixture of short dsRNA molecules called siRNA. In mammalian cells, thesiRNAs produced by Dicer are about 21 base-pairs (bp) in length. Thenthe siRNA join an RNase complex, RISC (RNA-induced silencing complex),which acts on the cognate mRNA and degrades it. RNA interference is alsoa valuable research tool, as double strand siRNA of 19 to 23 bp may beused to selectively and robustly induce suppression of specific genes ofinterest. The major interest of this approach is the specificity, as ansiRNA is able to discriminate two sequences even when differing by onlya single nucleotide.

The present inventors used said specificity of siRNA to specificallyinhibit one allele of a heterozygous DNM2 gene. Consequently, in thatparticular case, the siRNA is called an “allele specific siRNA”(AS-siRNA).

By AS-siRNA is meant any siRNA able to specifically silence only oneallele of a targeted gene, an allele being one of several alternativeforms of a gene occupying a given locus on a chromosome. “Genesilencing” refers to the suppression or reduction of gene expression.Gene silencing may be mediated through processes that affecttranscription and/or through processes that affect post-transcriptionalmechanisms. In some embodiments, gene silencing occurs when siRNAinitiates the degradation of the mRNA of the gene in a sequence-specificmanner via RNA interference. Thus, a gene includes coding sequencesand/or the regulatory sequences required for expression. For example,“gene” refers to a nucleic acid fragment that expresses mRNA, functionalRNA, or specific protein, including regulatory sequences. “Genes” alsoinclude nonexpressed DNA segments that may, for example, formrecognition sequences for other proteins.

In the context of the invention, the gene is the DNM2 gene, coding forDynamin 2 protein. Thus, an AS-siRNA of the invention specificallysilences one allele of the DNM2 gene, which is a variant form of theDNM2 gene.

In the context of the present invention, the DNM2 gene is a heterozygousDNM2 gene. A heterozygous DNM2 gene is a DNM2 gene present in aheterozygous state in a cell. By “heterozygous” is meant that a givenchromosomal locus has two different alleles. Diploid organisms such ashumans contain two copies of each chromosome (one maternal and onepaternal chromosome), that are called homologous chromosomes. Therefore,each homologous chromosome carries one allele of a given gene. A diploidorganism is heterozygous when said two alleles of a given gene aredifferent in respect to a given variation or polymorphism.

In the context of the invention, a cell or an organism is heterozygousin respect to the DNM2 gene wherein the DNM2 gene is present in aheterozygous state, that is to say wherein the two alleles of DNM2 geneare different in respect to a given variation or polymorphism.

In one embodiment of the invention, heterozygous refers to a genotype inwhich one allele has a wild-type DNM2 sequence and the other allele hasa sequence encoding a DNM2 variant. In particular, the sequence encodinga DNM2 variant comprises a mutation that is not present in the wild-typesequence.

In a first preferred embodiment, the DNM2 gene is heterozygous for thepresence of a disease-causing mutation. In this embodiment, the AS-siRNAof the invention targets the allele of the DNM2 gene comprising saiddisease-causing mutation.

In a second preferred embodiment, the DNM2 gene is heterozygous for thepresence of a non-pathological polymorphism. In this second embodiment,the AS-siRNA of the invention targets only one of the allele of the DNM2gene, comprising or not said non-pathological polymorphism.

In what follows, those two preferred embodiments are separatelydescribed.

AS-siRNA Targeting the Allele of the DNM2 Gene Comprising aDisease-Causing Mutation

DNM2 Gene Comprising a Disease-Causing Mutation

In one embodiment of the invention, the DNM2 gene is heterozygous forthe presence of a disease-causing mutation. Therefore, the DNM2 gene ispresent in two different forms corresponding to the two alleles: oneDNM2 allele is a “wild type allele” whereas the other is a “mutantallele”. In this embodiment, the AS-siRNA of the invention specificallytargets and silences the allele of the DNM2 gene comprising saiddisease-causing mutation without targeting the wild type allele. TheAS-siRNA is thus able to silence the expression of Dynamin 2 mRNA andDynamin 2 protein derived from the mutant allele without affecting theexpression of mRNA and protein derived from the wild type allele.

In particular, the disease-causing mutation could be any deletion,insertion or substitution of nucleotide(s) within the DNM2 gene which isresponsible for a pathology or which is correlated to a pathology. In apreferred embodiment, the disease-causing mutation is a dominantmutation. By dominant mutation is meant any mutation that leads to adominant allele. By “dominant allele” is meant an allele that exerts itseffect on phenotype over the presence of a recessive allele of the samegene. The terms dominant and recessive alleles are defined relative toone another, and are not absolute. In other words, the phenotypicconsequences of a dominant mutation are observed in a heterozygousindividual carrying one mutant allele and one wild type allele.Recessive alleles only show their effect if the individual has twocopies of the mutated allele (also known as being homozygous) or twodifferent mutated alleles (also known as composite heterozygosity).

In a particular embodiment, the dominant mutation is a gain-of-functionmutation. A gain-of-function mutation is defined as a mutation thatconfers new or enhanced activity on a protein. A gain-of-functionmutation is a type of mutation in which the altered gene productpossesses a new molecular function or a new pattern of gene expression.Consequently, the disease-causing mutation within the DNM2 leads to again-of-function of Dynamin 2 protein.

In a particular embodiment, the dominant mutation is a loss-of-functionmutation by dominant negative effect. A loss-of-function mutation isdefined as a mutation that results in the loss or reduction of thenormal activity of a protein. Dominant-negative effect is defined as theproduct of a mutated allele alters the function of the product from thewild-type allele. This occurs, for example, when oligomerization isrequired for normal function on a protein and when the mutated proteinis able to oligomerize with the wild-type protein. Consequently, thedisease-causing mutation within the DNM2 leads to a loss-of-function ofthe wild-type Dynamin 2 protein due to the presence of the mutatedDynamin 2 protein.

In another particular embodiment, the DNM2 gene which is heterozygousfor a disease-causing mutation is not haploinsufficient.Haploinsufficiency occurs when one copy of a gene is inactivated ordeleted and the remaining functional copy of the gene is not adequate toproduce sufficient amount of the gene product to preserve normalfunction. In other words, the wild type DNM2 allele of the invention isable to preserve normal function, following the silencing of the mutantallele by AS-siRNA of the invention. Therefore, the present inventionpreferably relates to autosomal dominant disease in which there is nohaploinsufficiency.

In a particular embodiment, the dominant mutation within the DNM2 geneleads to an autosomal dominant disease. An autosomal dominant disease isa disease wherein the individual has one copy of a mutant gene and onenormal gene on a pair of autosomal chromosomes (autosomal chromosomebeing any chromosome which is not a sex chromosome). An individual withautosomal dominant diseases has 50% chance of passing the mutant geneand therefore the disorder on to each of its children.

In one preferred embodiment, the autosomal dominant disease is selectedfrom Autosomal Dominant Centronuclear Myopathy (AD-CNM), T-cell acutelymphoblastic leukemia, Charcot-Marie-Tooth disease (CMT) or HereditarySpastic Paraplegia (HSP). Preferably, the autosomal dominant disease isAutosomal Dominant Centronuclear Myopathy (AD-CNM).

In another embodiment, the disease-causing mutation within the DNM2 geneis responsible for or correlated to a disease selected from AutosomalDominant Centronuclear Myopathy (AD-CNM), T-cell acute lymphoblasticleukemia, Charcot-Marie-Tooth disease (CMT) or Hereditary SpasticParaplegia (HSP). Preferably, the disease-causing mutation within theDNM2 gene is responsible for or correlated to Autosomal DominantCentronuclear Myopathy (AD-CNM).

In a preferred embodiment, the disease-causing mutation within the DNM2gene is a substitution of a single nucleotide, more preferably thedisease-causing mutation is a missense mutation. By missense mutation ismeant a point mutation in which a single nucleotide change results in acodon coding for a different amino acid. In a particular embodiment, thetarget allele encodes a protein with at least one mutation associated toAD-CNM selected in the group consisting of: p.E368K, p.E368Q, p.R369W,p.R369Q, p.V375G, p.R465W, p.R522H, p.R522C, p.R523G, p.E540K, p.E560K,p.D614N, p.A618T, p.A618D, p.S619L, p.S619W, p.L621P, p.624ins-1G,p.V625del, p.P627H, p.P627R, p.KDQ629-631de1-ins, p.P647R and p.E650K orwith at least one mutation associated to CMT selected in the groupconsisting of p.G358R, p.G537C, p.K554fs, p.D555_E557del, p.K559del,p.K562E, p.K562del, p.L570H, p.M580T, and p.859-860de1. For these twogroups of mutations, amino-acids are numbered relative to the human DNM2isoform 1 (NP_001005360.1). In a more preferred embodiment, the DNM2gene is heterozygous for the presence of a missense mutation selected inthe group consisting of: c.1393C>T; c.1105C>T; c.1106G>A; c.1393C>T;c.1856C>T or c.1948G>A, respectively responsible for the followingsubstitution in the DNM2 protein sequence: p.R465W, p.R369W, p.R369Q,p.R522H, p.S619L, and p.E650K. In an even more preferred embodiment, theDNM2 gene is heterozygous for the presence of the c.1393C>T mutation,responsible for the p.R465W substitution in the DNM2 protein sequence.

AS-siRNA

The AS-siRNA of the invention is able to silence the mutant allele ofDNM2 gene, by hybridizing specifically to the gene transcript (messengerRNA or mRNA) derived from said mutant allele of DNM2 gene. The AS-siRNAof the invention is therefore complementary to a mRNA derived from saidmutant allele of DNM2 and binds to said mRNA by base pairing. The term“complementary” refers to the ability of polynucleotides to form basepairs with another polynucleotide molecule. Base pairs are typicallyformed by hydrogen bonds between nucleotide units in antiparallelpolynucleotide strands. Preferably, the degree of complementaritybetween the AS-siRNA according to the invention and the target mRNA isequal to about 100%.

In a particular embodiment, the AS-siRNA of the invention targets aregion of the DNM2 gene transcript comprising said disease-causingmutation. Accordingly, the AS-siRNA of the invention is complementary toa sequence of the mRNA comprising said disease-causing mutation. Thespecificity of siRNA allows discriminating two sequences, even whendiffering by a single nucleotide. This property allows the AS-siRNA ofthe invention targeting disease-causing mutations, such as mutationsresulting to single nucleotide substitution.

In one embodiment of the invention, the AS-siRNA is of 19-23 base pairsin length, such as 19, 20, 21, 22 or 23. In a preferred particularembodiment, the AS-siRNA of the invention is of 19 base pairs in length.

In a particular embodiment, the AS-siRNA of the invention containsnucleotide overhangs on 3′ end of each strand. In a more particularembodiment, the AS-siRNA of the invention contains dinucleotideoverhangs made of two deoxythymidines (dTdT) on 3′ end of each strand.

In some embodiments, AS-siRNA of the invention is fully complementary tothe mutant DNM2 mRNA sequence comprising said disease-causing mutation(encoded by the mutant allele). On the other hand, the AS-siRNA of theinvention is complementary to the wild type DNM2 mRNA sequence (encodedby the wild type allele), except for a single nucleotide mismatch, at aposition wherein the nucleotide is not mutated, when compared to themutant mRNA. This mismatch enables the AS-siRNA to specifically targetand silence the mutant allele, while preserving the wild type allele.

In a particular embodiment, the DNM2 gene is heterozygous for thepresence of the c.1393C>T mutation, responsible for the p.R465Wsubstitution in the DNM2 protein sequence, and the AS-siRNA of theinvention is of 19 base pairs in length. Accordingly, the position ofsaid mismatch could be in any of the 19 possible positions within theAS-siRNA.

In a preferred embodiment, the position of the mismatch is located atposition N9, N10, N11, N12, N15 or N16 from 5′ end of the sense strandof AS-siRNA. More preferably, the position of the mismatch is located atposition N9 or N10, and even more preferably at position N9. By “sensestrand” is meant the strand of the AS-siRNA which has the same sequenceas the targeted mutant allele comprising said disease-causing mutation.Therefore, the other strand of AS-siRNA is called “anti-sense” becauseits sequence is complementary to the targeted mutant DNM2 mRNA, which iscalled the “sense” sequence (so that a sense segment of mRNA“5′-AAGGUC-3′” would be hybridized by the anti-sense mRNA segment“3′-UUCCAG-5′”).

By “position N9 from 5′ end of the sense strand” is meant the ninthnucleotide from the 5′ end of the sense strand (which has the samesequence as the target sequence). Position N9 from 5′ end of the sensestrand corresponds to position N11 from the 5′ of the antisense strand,when the siRNA is of 19 base pairs in length.

In a particular embodiment, the invention relates to an AS-siRNA of19-23 base pairs in length wherein the sense strand comprises a sequenceselected from the group consisting of: SEQ ID NO:5 to SEQ ID NO:10,preferably SEQ ID NO:5 or SEQ ID NO:6, and more preferably SEQ ID NO:5.

In a more particular embodiment, the invention relates to an AS-siRNA of19-23 base pairs in length wherein the sense strand consists in asequence selected from the group consisting of: SEQ ID NO:5 to SEQ IDNO:10, preferably SEQ ID NO:5 or SEQ ID NO:6, and more preferably SEQ IDNO:5.

In a particular embodiment, the AS-siRNA of the invention is used toreduce expression of total DNM2 mRNA and/or DNM2 protein by 20-60%, suchas 20, 30, 40, 50 or 60%. Preferably, the AS-siRNA of the invention isused to reduce expression of total DNM2 mRNA and/or DNM2 protein byabout 50%. By «about» is meant a value of + or −10%, preferably + or−5%. For example, about 50% means from 45 to 55%, preferably from 47.5to 52.5%.

As-siRNA Targeting the Allele of DNM2 Gene Comprising a Non-PathologicalPolymorphism

DNM2 Gene Comprising a Non-Pathological Polymorphism

Another aspect of the invention relates to an AS-siRNA able to silencethe expression of only one allele of a heterozygous DNM2 gene in a cell,wherein the DNM2 gene is heterozygous for the presence of anon-pathological polymorphism.

In a particular embodiment, the DNM2 gene comprises a commonheterozygous non-pathological polymorphism. “Common heterozygous nonpathological polymorphism” refers to a polymorphism with highheterozygous frequency that is to say that is frequent in the populationat heterozygous state. By “frequent” is meant a polymorphism which isfound at heterozygous state in at least 20%, 30%, 40% of generalpopulation, preferably at least 40%.

By “non-pathological polymorphism” is meant a variation in the nucleicacid sequence of a gene that is not associated with a disease. As such,according to the present invention, a non-pathological polymorphismcorresponds to a sequence variation in a gene that, when consideredindependently of other sequence modifications, is not by itselfassociated to a pathology. For the sake of clarity, if anon-pathological polymorphism is heterozygous in a cell, it means thatboth polymorphisms are considered non-pathological, if consideredindependently of other sequence variations that might occur on the samegene. Non-pathological polymorphisms may include variations in codingand non-coding regions. Furthermore, non-pathological polymorphismsinclude nucleotide substitutions, deletions, and/or additions, includingthose that result in missense and nonsense mutations which do not leadto a pathology. Preferably, the non-pathological polymorphism is asingle nucleotide substitution.

By targeting heterozygous common polymorphisms, the AS-siRNA of theinvention can be used to reduce the expression of DNM2 protein,especially when overexpression of DNM2 in absence of mutation isassociated with a pathological condition. For example, overexpression ofDNM2 protein, in absence of mutation is correlated to X-linkedmyotubular myopathy or cancer such as prostate cancer and pancreaticcancer. Thus, the invention relates to an AS-siRNA wherein the AS-siRNAtargets a DNM2 allele comprising a non-pathological polymorphism.

In another embodiment, the DNM2 allele comprising a non-pathologicalpolymorphism is on the same allele as a heterozygous disease-causingmutation. By targeting heterozygous common polymorphisms rather thaneach specific disease-causing mutation, a single AS-siRNA can be used toinhibit expression of more than one disease-causing mutation in morethan one patient. Accordingly, in one embodiment, the targeted versionof the heterozygous non-pathological polymorphism is present on the sameallele as said disease-causing mutation and is absent on the wild typeallele which harbors the other version of the polymorphism. In otherwords, targeting a heterozygous non-pathological polymorphism allowsdifferentiating mutant and wild-type DNM2 alleles. Said disease-causingmutation can be any heterozygous mutation within the DNM2 generesponsible for or associated to a disease. For example, thedisease-causing mutation within the DNM2 gene is responsible for orcorrelated to a disease selected from Autosomal Dominant CentronuclearMyopathy (AD-CNM), T-cell acute lymphoblastic leukemia,Charcot-Marie-Tooth disease (CMT) or Hereditary Spastic Paraplegia(HSP). Preferably, the disease-causing mutation within the DNM2 gene isresponsible for or correlated to Autosomal Dominant CentronuclearMyopathy (AD-CNM). In a more preferred embodiment, the DNM2 gene isheterozygous for the presence of a missense mutation selected in thegroup consisting of: c.1393C>T; c.1105C>T; c.1106G>A; c.1393C>T;c.1856C>T or c.1948G>A, respectively responsible for the followingsubstitution in the DNM2 protein sequence: p.R465W, p.R369W, p.R369Q,p.R522H, p.S619L, and p.E650K. In an even more preferred embodiment, theDNM2 gene is heterozygous for the presence of the c.1393C>T mutation,responsible for the p.R465W substitution in the DNM2 protein sequence.

In a particular embodiment, the heterozygous non-pathologicalpolymorphism is rs2229920 (C or T) or rs12461992 (A or T), preferablyrs2229920 (C or T).

AS-siRNA

The AS-siRNA of the invention is able to silence only one allele of DNM2gene comprising a heterozygous non-pathological polymorphism, byhybridizing specifically to the gene transcript (messenger RNA or mRNA)derived from said allele of DNM2 gene. AS-siRNA of the invention istherefore complementary to mRNA derived from said allele of DNM2 andbinds to said mRNA by base pairing. The term “complementary” refers tothe ability of polynucleotides to form base pairs with anotherpolynucleotide molecule. Base pairs are typically formed by hydrogenbonds between nucleotide units in antiparallel polynucleotide strands.Preferably, the degree of complementarity between the AS-siRNA accordingto the invention and the target mRNA is equal to about 100%.

In a particular embodiment, AS-siRNA of the invention targets a regionof the DNM2 gene transcript comprising said non-pathologicalpolymorphism. Accordingly, the AS-siRNA of the invention iscomplementary to a sequence of the mRNA comprising said non-pathologicalpolymorphism. The specificity of siRNA allows discriminating twosequences, even when differing by a single nucleotide. This propertyallows the AS-siRNA of the invention targeting polymorphism resulting tosingle nucleotide substitution.

In one particular embodiment, the target allele (and consequently thetarget mRNA) could be arbitrarily chosen, in absence of disease-causingmutation, in order to reduce the level of DNM2 mRNA and/or protein. Inparticular, the AS-siRNA of the invention could target the mRNA carryingor not said non-pathological polymorphism, when the goal is only toreduce the overall level of DNM2 mRNA or DNM2 protein. For example, theAS-siRNA of the invention could target anyone of the DNM2 allelecarrying one of the two version of the heterozygous non-pathologicalpolymorphism, wherein DNM2 is overexpressed in a cell, for example inX-linked myotubular myopathy or cancer such as prostate cancer andpancreatic cancer.

In another particular embodiment, the targeted version of thenon-pathological polymorphism is present on the same allele as adisease-causing mutation. Therefore, the AS-siRNA of the inventiontargets and silences only the allele carrying said targeted version ofthe polymorphism and said disease-causing mutation. This particularembodiment requires prior confirming the location of the disease-causingmutation.

In one embodiment of the invention, the AS-siRNA is of 19-23 base pairsin length, such as 19, 20, 21, 22 or 23. In a preferred particularembodiment, the AS-siRNA of the invention is of 19 base pairs in length.

In a particular embodiment, the AS-siRNA of the invention containsnucleotide overhangs on 3′ end of each strand. In a more particularembodiment, the AS-siRNA of the invention contains dinucleotideoverhangs made of two deoxythymidines (dTdT) on 3′ end of each strand.

In some embodiments, the AS-siRNA of the invention is fullycomplementary to the DNM2 mRNA sequence comprising said targeted versionof non-pathological polymorphism. On the other hand, the AS-siRNA of theinvention is complementary to the DNM2 mRNA sequence that does notcomprise said polymorphism (encoded by the other allele), except for onesingle nucleotide mismatch, at a position wherein the polymorphism isabsent. This mismatch enables the AS-siRNA to specifically target andsilence only the allele carrying said polymorphism, while preserving theother allele.

In a particular embodiment, the DNM2 gene is heterozygous for thepresence of the non-pathological polymorphism rs2229920 (C or T), whichis found in 40% of individuals at heterozygous state. Therefore, theinvention relates to an AS-siRNA that targets either the sequencecomprising a C or the sequence comprising a T (corresponding to U inmRNA sequence).

In a preferred embodiment, the AS-siRNA of the invention targets thenon-pathological polymorphism rs2229920 (C or T) and is of 19 base pairsin length. Accordingly, the position of the said mismatch could be inany of the 19 possible positions within the AS-siRNA.

In a preferred embodiment, the position of the mismatch is located atposition N16 or N17 from 5′ end of the sense strand of AS-siRNA. Morepreferably, the position of the mismatch is located at position N17. By“sense strand” is meant the strand of the AS-siRNA which has the samesequence as the targeted allele comprising said non-pathologicalpolymorphism. Therefore, the other strand of AS-siRNA is called“anti-sense” because its sequence is complementary to the targeted DNM2mRNA, which is called the “sense” sequence (so that a sense segment ofmRNA “5′-AAGGUC-3′” would be blocked by the anti-sense mRNA segment“3′-UUCCAG-5′”).

By “position N17 from 5′ end of the sense strand” is meant theseventeenth nucleotide from the 5′ end of the sense strand (which hasthe same sequence as the target sequence). Position N17 from 5′ end ofthe sense strand corresponds to position N3 from the 3′ of the antisensestrand, wherein the siRNA is of 19 base pairs in length.

In a particular embodiment, the invention relates to an AS-siRNA of19-23 base pairs in length, wherein the sense strand comprises asequence selected from the group consisting of: SEQ ID NO:1 to SEQ IDNO:4, preferably SEQ ID NO:1 or SEQ ID NO:2.

In a more particular embodiment, the invention relates to an AS-siRNA of19-23 base pairs in length, wherein the sense strand consists of asequence selected from the group consisting of: SEQ ID NO:1 to SEQ IDNO:4, preferably SEQ ID NO:1 or SEQ ID NO:2.

In a particular embodiment, the AS-siRNA of the invention is used toreduce expression of DNM2 mRNA and/or DNM2 protein by 20-60%, such as20, 30, 40, 50 or 60%. Preferably, the AS-siRNA of the invention is usedto reduce expression of DNM2 mRNA and/or DNM2 protein by about 50%. By«about» is meant a value of + or −10%, preferably + or −5%. For example,about 50% means from 45 to 55%, preferably from 47.5 to 52.5%.

Methods and Uses of AS-siRNA

The present invention contemplates various ways of reaching the targetmRNA with AS-siRNA of the invention. The AS-siRNA may be administered tothe cell as isolated oligonucleotide, either directly or usingtransfection reagents such as lipidic derivatives, liposomes, calciumphosphate, nanoparticles, microinjection or electroporation.

In another embodiment, the present invention contemplates introducingthe AS-siRNA into the cell in the form of a vector. Thus, another aspectof the present invention relates to a vector encoding the AS-siRNA ofthe invention. The vector may in particular be a plasmid or a viralvector. Representative viral vectors useful in the practice of theinvention include, without limitation, a vector derived from adenovirus,retrovirus, in particular lentivirus, poxviruses, herpes simplex virus Iand adeno-associated virus (AAV). In a particular embodiment, the vectoris an AAV1, AAV8 or AAV9 vector. Selection of the appropriate viralvector will of course depend on the targeted cell and the virus tropism.In a particular embodiment, targeted cells are muscle cells, but viralvectors with broad tropism, including in particular the muscle tropism,may also be implemented. In a particular embodiment, an AAV1 vector isimplemented, for example for use in intramuscular injections. In anotherembodiment, the vector is to be administered via the systemic route (forexample via the intravascular or intraarterial route), and the vector isan AAV8 or AAV9 vector.

In a particular embodiment, the invention also relates to shRNA (shorthairpin RNA) corresponding to the AS-siRNA of the invention, with afurther tight hairpin turn. The shRNA hairpin structure is then cleavedby the cellular machinery into siRNA. A further aspect of the inventionrelates to a vector encoding shRNA corresponding to AS-siRNA of theinvention. In another aspect, the invention also relates to a targetcell comprising an AS-siRNA of the invention or which is transfected ortransduced with a vector of the invention. For example, the target cellmay be selected from: a muscle cell (or a cell of the muscle lineage),such as myoblast, for example a patient-derived myoblast, or afibroblast such as a patient-derived fibroblast.

In another aspect, the present invention relates to an in vitro methodfor silencing the expression of the mutated allele of DNM2 gene withoutsilencing the expression of the wild type allele of the DNM2 gene in atarget cell, such as a muscle target cell (for example a muscle cell,such as a myoblast, in particular a patient-derived myoblast),comprising introducing in said target cell an AS-siRNA or a vector ofthe invention.

In another aspect, the present invention relates to an in vitro methodfor silencing the expression of one allele of DNM2 gene carrying aheterozygous non-pathological polymorphism without silencing theexpression of the other allele of the DNM2 gene in a target cell,comprising introducing in said target cell an AS-siRNA or a vector ofthe invention.

In a further aspect, the present invention relates to an AS-siRNA, avector or a cell of the invention for use in a method for treating adisease induced by a disease-causing mutation in the DNM2 gene.Preferably, an AS-siRNA, a vector or a cell of the invention are used ina method for treating centronuclear myopathy (such as autosomal dominantcentronuclear myopathy), T-cell acute lymphoblastic leukemia,Charcot-Marie-Tooth disease (CMT) or Hereditary Spastic Paraplegia(HSP). More preferably, an AS-siRNA, a vector or a cell of the inventionare used in a method for treating autosomal dominant centronuclearmyopathy.

In another aspect, the present invention relates to an AS-siRNA, avector or a cell of the invention for use in a method for treating amuscular dystrophy such as Duchenne muscular dystrophy.

In another aspect, the present invention relates to an AS-siRNA, avector or a cell of the invention for use in a method for treating adisease associated with overexpression of dynamin 2, preferably fortreating X-linked myotubular myopathy, or cancer such as prostate cancerand pancreatic cancer.

As used herein, the term “treating” and “treatment” refers toadministering to a subject an effective amount of an AS-siRNA of theinvention so that the subject has a reduction in at least one symptom ofthe disease or an improvement in the disease, for example, beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, alleviation ofone or more symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. Treating can refer to prolonging survival as compared toexpected survival if not receiving treatment. Alternatively, treatmentis “effective” if the progression of a disease is reduced or halted.“Treatment” can also mean prolonging survival as compared to expectedsurvival if not receiving treatment. Those in need of treatment includethose already diagnosed with a DNM2 associated disorder, as well asthose likely to develop such a disorder due to genetic susceptibility orother factors. As used herein, the term “treating” and “treatment” alsorefers the prevention of a disease or disorder, which means delaying orpreventing the onset of such disease or disorder.

The AS-siRNA of the invention, the vector or the cell according to theinvention can be formulated and administered to treat any disease causedby a heterozygous mutation in the DNM2 gene or caused by overexpressionof DNM2, preferably to treat autosomal dominant centronuclear myopathy,T-cell acute lymphoblastic leukemia, Charcot-Marie-Tooth disease,Hereditary Spastic Paraplegia, X-linked myotubular myopathy, or cancersuch as prostate cancer and pancreatic cancer. AS-siRNA of theinvention, the vector or the cell according to the invention areformulated by any means that produces contact of the AS-siRNA with itssite of action in the subject in need thereof.

The present invention also provides pharmaceutical compositionscomprising the AS-siRNA of the invention, the vector or the cellaccording to the invention. Such compositions comprise a therapeuticallyeffective amount of the therapeutic (the AS-siRNA, vector or cell of theinvention), and a pharmaceutically acceptable carrier. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. or European Pharmacopeia or other generally recognized pharmacopeiafor use in animals, and humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas saline solution, water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. Physiological saline solution is apreferred carrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc,sodium chloride, dried skim milk, glycerol, propylene glycol, water,ethanol and the like.

The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents. These compositions cantake the form of solutions, suspensions, emulsions, tablets, pills,capsules, powders, sustained-release formulations and the like. Oralformulation can include standard carriers such as pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate, etc. Examples of suitable pharmaceuticalcarriers are described in “Remington's Pharmaceutical Sciences” by E. W.Martin. Such compositions will contain a therapeutically effectiveamount of the therapeutic, preferably in purified form, together with asuitable amount of carrier so as to provide the form for properadministration to the subject.

The pharmaceutical composition is adapted for any type of administrationto a mammal, in particular a human being and is formulated in accordancewith routine procedures. The composition is formulated by using suitableconventional pharmaceutical carrier, diluent and/or excipient.Administration of the composition may be via any common route so long asthe target tissue is available via that route.

The amount of the therapeutic of the invention which will be effectivein the treatment of a nucleotide repeat expansion can be determined bystandard clinical techniques. In addition, in vivo and/or in vitroassays may optionally be employed to help predict optimal dosage ranges.The precise dose to be employed in the formulation will also depend onthe route of administration, and the seriousness of the disease, andshould be decided according to the judgment of the practitioner and eachpatient's circumstances. The dosage of the AS-siRNA, the vector or thecell administered to the subject in need thereof will vary based onseveral factors including, without limitation, the route ofadministration, the subject's age or the level of expression necessaryto obtain the required therapeutic effect. One skilled in the art canreadily determine, based on its knowledge in this field, the dosagerange required based on these factors and others.

EXAMPLES

AS-siRNA Targeting the Allele of the DNM2 Gene Comprising aDisease-Causing Mutation

Materials and Methods

Cell Cultures

Mouse Embryonic Fibroblasts (MEF) were prepared from 13.5 day-oldembryos. Cells were cultured at 37° C. (5% CO2) in Dulbecco's modifiedEagle's medium (DMEM) containing 10% foetal calf serum (FCS)supplemented with penicillin, streptomycin, L-glutamate, and sodiumpyruvate. Experiments were performed on MEFs in primary cultures, i.e. 2or 3 passages after embryo dissection. Human skin fibroblasts fromhealthy control subjects (C1 and C2) and from one DNM2-linked CNMpatient harbouring the p.R465W mutation were cultured using the samemedium. Experiments were performed on immortalized cell population aftertransduction with lentivirus expressing human telomerase reversetranscriptase (hTERT). For transfection, cells were grown to 70%confluency and transfected with siRNA using JetPrime transfectionreagent according to the manufacturer's protocol (Polyplus Transfection,France). Concentration of siRNA for each experiment are indicated infigure legend. Scramble siRNA (Eurogentec, Belgium) was used as control.Cells were harvested 48 h later for RNA and protein extraction orimmunohistochemistry.

Total RNA Extraction and cDNA Analysis

Total RNA was isolated using RNA easy or RNA easy Fibrous tissue minikits (Qiagen, France) for cells and muscles, respectively, according tothe manufacturer's protocol. Cells or tissue sections were passedthrough a 22G syringe several times for disruption in the lysis buffer.Total RNA (1 μg) was submitted to reverse transcription using theSuperscript III reverse transcriptase kit (Life Technologies) usinghexamer primers. cDNA were amplified by PCR under the followingconditions: 96° C. for 5 min, cycles of 30 s at 96° C., 30 s at theappropriate temperature (58 to 62° C.), 30 s to 1 min at 72° C., and afinal step of 7 min at 72° C. Twenty three cycles were performed toamplify Gapdh, 28 for semi-quantitative analysis of Dnm2, 30 forrestriction enzyme digestion of the human and murine Dnm2 amplicons, and30 for genotyping and for the other PCRs. Sequences of all the primersused are indicated in the following table:

Table: Primers used in this study Target Sequence (5′-3′) ApplicationmGapdh F ACCACAGTCCATGCCATCAC Semi-quantitative analysis in RTCCACCACCCTGTTGCTGTA mouse hHPRT F ACCCCACGAAGTGTTGGATASemi-quantitative analysis in R AAGCAGATGGCCACAGAACT human cells mDnm2 FGGTGGTCAAGCTGAAAGAG Semi-quantitative analysis in MEF RGCTGTCAGCACGAACCAGTA and EcoNI digestion profile mDnm1 FAGATGGAGCGAATTGTGACC Semi-quantitative analysis in RGAATGACCTGGTTCCCTGAA mouse mDnm3 F ATGCTCCGAATGTACCAAGCSemi-quantitative analysis in R GAGGGGAGCACTTATCGTCA mouse mDnm2speMut FAATTGTCACCACCTACATCA Specific amplification of the RGGTTTGTGTTGATGTACGACTGC mutated Dnm2 in mouse mDnm2speWT FAATTGTCACCACCTACATCT Specific amplification of the WT RGGTTTGTGTTGATGTACGACTGC Dnm2 in mouse mDnm2g F CTGCGAGAGGAGACCGAGCGenotyping (knock-in mice) R GCTGAGCACTGGAGAGTGTATGG hDnm2 FGAAAAAGCAGGTCGTCAAGC Semi-quantitative analysis in R ATTGGGGATGGCTCTCTThuman cells and PfoI digestion profile Legend: F: Forward. R: Reverse.Dnm2: Dynamin 2. Gapdh: Glyceraldehyde 3-phosphate dehydrogenase.

The half of the PCR products was used for restriction enzyme digestionwith 2U of EcoNI (New England Biolabs, France) or PfoI (ThermoFisherScientific, France) for 2 h at 37° C. Image acquisition of PCR productsafter agarose gel electrophoresis was performed using G-box (Ozyme,France) and associated-signal was quantified using ImageJ Software (NIH;http://rsbweb.nih.gov/ij). DNA sequencing was performed on 20 ng DNA/100bp with 5 pmol of primers (Eurofins, France).

Protein Extraction and Western Blot

Cell pellets and frozen Tibialis anterior muscles (TA) were homogenizedin lysis buffer containing 50 mM of Tris-HCl pH 7.5, 150 mM NaCl, 1 mMEDTA, NP40 1%) supplemented with protease inhibitor cocktail 1%(Sigma-Aldrich, France). In addition, Tibialis anterior muscles weremechanically homogenized in the lysis buffer using Fastprep LysingMatrix D and Fastprep apparatus (MP Biomedical, France). Aftercentrifugation (14,000 g, 4° C., 15 min), protein concentration in thesupernatant was determined with the BCA Protein Assay Kit (ThermoScientific Pierce, France). Twenty μg of protein were mixed with loadingbuffer (50 mM Tris-HCl, SDS 2%, glycerol 10%, β-mercaptoethanol 1% andbromophenol blue) and denaturated at 90° C. for 5 min. Protein sampleswere separated on SDS-PAGE 10% and transferred onto PVDF membranes (0.45μm pore size, Life Technologies) overnight at 100 mA at 4° C. Membraneswere blocked for 1 h at room temperature in PBS containing non-fat drymilk 5% and Tween20 0.1% and then exposed to the following primaryantibodies: rabbit polyclonal anti-Dynamin 2 (Abcam ab3457, UnitedKingdom) and rabbit polyclonal anti-GAPDH (Santa Cruz, France) inPBS-Tween20 0.1%, milk 1% overnight at 4° C. Membranes were rinsed inPBS-Tween20 0.1% and incubated 1 h with secondary horseradishperoxidase-conjugated antibodies (anti-rabbit from JacksonImmunoResearch, United Kingdom) in PBS-Tween20 0.1%. Chemiluminescencewas detected using ECL detection Kit (Merck-Millipore, Germany) in G-Box(Ozyme, France) and associated-signal quantification was performed usingImageJ software.

AAV Production and In Vivo AAV Injection

AAV2/1 pseudotyped vectors were prepared by transfection in 293 cellsusing the pSMD2-Dnm2-PTM plasmid, the pXX6 plasmid coding for theadenoviral sequences essential for AAV production, and the pRepCApplasmid coding for AAV1 capsid. Vector particles were purified oniodixanol gradient and concentrated on Amicon Ultra-15 100K columns(Merck-Millipore). The particle titer (number of viral genomes (vg)/ml)was determined by quantitative PCR. Wild-type and heterozygousKI-Dnm2^(R465W) mice at 1 month of age were injected under isofluraneanesthesia. Two intramuscular injections (30 μl/TA, within 24 hinterval) of AAV-sh9, AAV-sh10, or AAV-nosh (negative control withoutshRNA sequence in the viral genome) were performed in 2 TA using 29Gneedle at 10¹¹ vg/muscle. Animal studies conform to the French laws andregulations concerning the use of animals for research and were approvedby an external Ethical committee (approval n°00351.02 delivered by theFrench Ministry of Higher Education and Scientific Research).

Muscle Contractile Properties

The isometric contractile properties of TA muscles were studied in situon mice anesthetized with 60 mg/kg pentobarbital. The distal tendon ofthe TA muscle was attached to a lever arm of a servomotor system (305BDual-Mode Lever, Aurora Scientific). The sciatic nerve was stimulated bya bipolar silver electrode using a supramaximal (10 V) square wave pulseof 0.1 ms duration. Absolute maximal isometric tetanic force (P0) wasmeasured during isometric contractions in response to electricalstimulation (frequency of 25-150 Hz; train of stimulation of 500 ms).All isometric contraction measurements were made at optimal musclelength (L0) at which P0 was obtained. TA muscles were weighted andspecific force (sP0) was calculated by dividing P0 by muscle weight.

Histomorphological Analyses

Mice were sacrificed by cervical dislocation under isofluraneanesthesia. TA muscles were frozen in liquid nitrogen-cooled isopentane.Transverse sections of TA muscle (8 μm thick) were stained withhematoxylin and eosin (HE) and reduced nicotinamide adeninedinucleotide-tetrazolium reductase (NADH-TR) by standard methods. Lightmicroscopy were performed using an upright microscope (DMR, Leica) andimages were captured using a monochrome camera (DS-Ri1, Nikon) andNIS-Elements BR software (Nikon, France). For all imaging, exposuresettings were identical between compared samples and viewed at roomtemperature. Fiber size distribution was determined on TA musclesections immunocytochemically labelled with laminin by measuring Ferretdiameter using ImageJ software.

Immunocytochemistry

Cells and TA cryosections (8 μm thick) were fixed in paraformaldehyde 4%(15 min at room temperature). After washing in PBS, cells andcryosections were permeabilized in Triton X-100 0.5% in PBS for 10 minat room temperature and blocked in PBS-Triton X-100 0.1%, BSA 5% andDonkey serum 5% for 30 min. Samples were incubated with primaryantibodies: rabbit anti-C-terminal Dynamin 2 (Abcam ab3457) orrabbit-anti Laminin (Abcam ab11575) overnight at 4° C., in PBS withTriton X-100 0.1% and BSA 1%. After PBS-Triton X100 0.1% washes, sampleswere incubated with Donkey anti-rabbit Alexa 488 secondary antibody(Life Technologies, France) for 60 min at room temperature. The slideswere mounted with VECTASHIELD mounting medium (Vector Laboratories,United Kingdom). Images were acquired using either axiophot microscope(Zeiss) or confocal microscope (Olympus FV-1000).

Transferrin Uptake Assay

Two healthy control cell lines (C1 and C2) and one patient-derived cellline expressing the p.R465W mutation were used 48 h after transfectionwith siRNA. Cells were cultured in DMEM without FCS at 37° C. for 45min. Transferrin-AlexaFluor488 (Life Technologies, France) was added at40 μg/ml and cells were incubated at 37° C. for 15 min. Cells werewashed 3 times with PBS and fixed in paraformaldehyde 4% at roomtemperature for 15 min. Stacks of cell images (0.5 μm interval) weregained using a confocal Leica SP2 microscope. Fluorescent-positivesurface was quantified on stack projection using ImageJ software andnormalized to the total cell surface.

Results

Identification of Allele-Specific siRNA in Heterozygous Cells

The p.R465W DNM2 mutation was used to construct the KI-Dnm2^(R465W)mouse model. In mouse, this missense mutation corresponds to a singlepoint mutation A>T in exon 11. A screening for allele specific siRNAsilencing the mutated Dnm2 allele without affecting the WT one wasperformed in Mouse Embryonic Fibroblasts (MEF) cultured fromheterozygous (HTZ) KI-Dnm2 mice. Using a RT-PCR assay developed todiscriminate the WT and mutated allele after restriction enzymedigestion of amplicon, we assessed allele-specific properties of 12siRNA among the 19 possible siRNA (FIG. 1A). In FIG. 1A. siRNA sensestrands are represented. It is the other siRNA strand (the antisensestrand) that binds by complementarity to the target mutant mRNA). At lowconcentration (20 nM), scramble siRNA-transfected cells andnon-transfected cells show a Mutant/WT ratio equal to 1 in agreementwith similar expression of both WT and mutated alleles (FIG. 1B). Amongthe 12 assessed siRNA, 6 siRNA (si9, si10, si11, si12, si15, si16)exhibit allele-specific silencing properties as demonstrated bysignificantly reduced Mutant/WT ratio compared to scramblesiRNA-transfected control cells (FIG. 1B). Within those 6 siRNA, theposition of the mismatch is respectively located at position N9, N10,N11, N12, N15 or N16 from 5′ end of the sense strand of AS-siRNA.

In order to establish allele-specific silencing, further analyses werepursued for 2 of the most efficient siRNA, i.e. si9 and si10 at highconcentration (100 nM). After 48 h, total Dnm2-mRNA expression(WT+mutated) is reduced around 50% for both siRNA (FIG. 2A). Sequencingof the amplicons showed HTZ Dnm2 sequence in scramble-transfected cellsand only WT sequence in si9- and si10-transfected cells (FIG. 2B).Allele-specificity of the two siRNA against the mutated allele isdemonstrated by the measured mutant/WT ratio around 0.2 (FIG. 2C) and isconfirmed by quantification of the expression level of both Mutant andWT mRNA relative to the housekeeping Gapdh mRNA expression (Data notshown). At protein level, both si9 and si10 induced a decrease in Dnm2expression level around 50% as established by western blot (FIG. 2D)without modifying Dnm2 subcellular localization (Data not shown). Underthese conditions, si9 and si10 do not affect expression of Dnm1 and Dnm3transcripts (Data not shown). Altogether, these data validate si9 andsi10 as efficient allele-specific siRNA since both specificallyknock-down the mutated Dnm2 transcript without affecting the WTresulting to a remaining expression of the half of the Dnm2 transcriptand protein. The ability of 100 nM of si9 and si10 to specificallysilence the mutant allele was confirmed by RT-PCR in immortalized mousemyoblasts derived from HTZ KI-Dnm2 mice (FIG. 3).

Restoration of the Muscle Phenotype in KI-Dnm2 Mice

Muscle phenotype in TA muscle from HTZ KI-Dnm2 mice is fully establishedat 2 months of age and includes impairment of contractile properties,muscle atrophy due to decrease in fibre size, and morphologicalabnormalities on oxidative staining. Adeno-associated virus (AAV)vectors expressing shRNA corresponding to si9 and si10 and a control AAVwithout shRNA sequence (AAV-nosh) were constructed for in vivoevaluation. AAV were injected intramuscularly in TA muscles of HTZKI-Dnm2 mice at 1 month of age and muscle phenotype was investigated 3months later. Compared to WT TA, a significant decrease around 30% inmass, 40% in absolute force and 15% in specific force is present innosh-injected HTZ muscles (FIG. 4). Expression of sh9 fully restoredabsolute and specific force to the WT values. Muscle mass is alsolargely increased compared to noSh muscles (+30%) but remains slightlylower than WT muscle mass (−10%) in sh9-injected mice. Significantrestoration is also achieved by expression of sh10 by increasingabsolute and specific forces and muscle mass. (FIG. 4).

Expression level of Dnm2 transcript was quantified by RT-PCR showing asignificant decrease (−30%) in the Dnm2 content in sh9-expressing musclecompared to nosh values whereas statistical significance is not reachedin muscle expressing sh10 (FIG. 5A). In agreement, sequencing of Dnm2amplicons showed a HTZ sequence at the mutated nucleotide position inAAV-transduced muscles with a substantial reduction of the peakcorresponding to the mutated nucleotide with sh9 and to a lesser extentwith sh10 (FIG. 5B). Allele-specific silencing was evaluated byquantifying Mutant/WT ratio using RT-PCR and EcoNI digestion profile. Anexpected reduction of Mutant/WT ratio was achieved with values reachingaround 0.5 and 0.7 for sh9 and sh10, respectively (FIG. 5C). Specificsilencing of the mutated transcript achieved by sh9 was also shown byquantification of digested products corresponding to WT and Mutant mRNArelative to Gapdh expression and was confirmed by a second RT-PCR assayusing primers designed for specific amplification of either WT ormutated allele (data not shown).

The restoration of phenotype at histological level was furtherevaluated. When compared to WT muscle, HTZ TA muscles show a reductionof fibre size illustrated on HE staining and central accumulation ofoxidative material on DPNH oxidative staining (FIG. 5D). Fibre sizeappeared higher in sh9-expressing muscles and abnormalities were notvisible on DPNH staining. In contrast morphological abnormalities werestill present in sh10-expressing TA. Calculation of frequency of fibresize (FIG. 5E) confirm the total rescue of these parameters only in HTZTA muscles transduced by sh9-AAV. Altogether, these data demonstrate thecapability of sh9 to fully reverse muscle phenotype in mice after a3-month treatment whereas sh10 only induced a partial restoration underthe same conditions. However, optimisation of the amount of vector, timeof treatment or other adaptations well within the general knowledge ofthe skilled person may be implemented for improving this restoration.

Allele-Specific Silencing in Patient-Derived Cells

The Dnm2 target sequence for si9 and si10 shows 79% identity (15 out of19 bp) between mouse and human sequence. Human-specific si9 and si10were produced for in vitro evaluation in one patient-derived fibroblastcell line expressing the p.R465W mutation. Dnm2 mRNA content is reducedaround 50% in cells transfected with si9 for 48 h at 50 nM (FIG. 6A).Amplicon sequencing confirms disappearance of the mutated mRNA insi9-transfected fibroblasts compared to scramble- and si10-transfectedcells still expressing a mix of WT and mutated DNM2 (FIG. 6B). We used asemi-quantitative RT-PCR assay developed to discriminate the WT andmutated allele after PfoI digestion of amplicon. Using this assay,mutant/WT ratio is equal to 1 for non-transfected andscramble-transfected cells and significantly reduced to 0.8 by si10 and0.2 by si9 (FIG. 6C). Transfection of both si9 and si10 for 48 hresulted in reduction of DNM2 protein content as demonstrated bywestern-blot (FIG. 6D). Given that si9 exhibited all the expectedproperties of allele-specific siRNA in human cells, si9 was consequentlyfurther investigated at higher dose (100 nM). At this dose, expressionof DNM2 transcript was still reduced around 50% (FIG. 7A) andallele-specificity against the mutated DNM2 mRNA is maintained (FIG. 7B)without evident cell toxicity (FIG. 7C). Basic Local Alignment SearchTool (BLAST) used to identify potential off target for si9 (sense andantisense strands) showed SLC9A8 as the nearest sequence with 68%identity corresponding to complete identity on 13 consecutivenucleotides. We checked for potential si9-induced silencing of SLC9A8mRNA by RT-PCR 48 hours after transfection of siRNA at 100 mM. Comparedto Scramble siRNA, si9 does not affect expression of the SLC9A8transcript (data not shown).

si9 properties was finally evaluated by investigating functional rescuein patient-derived cells. Clathrin-mediated endocytosis is impaired infibroblast from patient. A fluorescent-transferrin uptake assay was usedto evaluate capability of si9 to restore normal endocytosis. Compared to2 healthy control cell lines, 15 minutes transferrin uptake is reducedin scramble-transfected cells from the CNM patient but achieved normalvalue in cells transfected with si9 for 48 hours before assay (FIG. 8A).Transfection of si9 in control cell lines does not impactclathrin-mediated endocytosis (FIG. 8B) in agreement with absence ofeffect on WT Dnm2 mRNA.

Discussion

Allele-specific silencing by RNAi benefits from outstanding specificityof RNA interference process mediated by siRNA able to discriminate twosequences, even when differing by a single nucleotide. This propertyqualified allele-specific RNAi to target dominant mutations resulting tosingle nucleotide substitution representing the majority of the 24 DNM2mutations identified in patients suffering from AD-CNM, especially forthe most frequent of them (30% of patients), i.e. the p.R465W mutation.The above results demonstrated the efficiency of allele-specific RNAiagainst the p.R465W mutation as therapeutic strategy for DNM2-relatedCNM through rescue animal model and in patient-derived cells.

In a context of dominant inherited disease in which WT and mutatedalleles are similarly expressed, allele-specific siRNA are expected toreduced expression of target mRNA and protein around 50% resulting fromsilencing of the mutated allele without affecting the WT. This is thecase in this study for si9 in murine and human cells.

As-siRNA Targeting the Allele of the DNM2 Gene Comprising aNon-Pathological Polymorphism

Materials and Methods

Cell Cultures

Human skin fibroblasts from one DNM2-linked CNM patient harbouring thep.R465W mutation were cultured at 37° C. (5% CO2) in Dulbecco's modifiedEagle's medium (DMEM) containing 10% foetal calf serum (FCS)supplemented with penicillin, streptomycin, L-glutamate, and sodiumpyruvate. Experiments were performed on immortalized cell populationafter transduction with lentivirus expressing human telomerase reversetranscriptase (hTERT). For transfection, cells were grown to 70%confluency and transfected with siRNA using JetPrime transfectionreagent according to the manufacturer's protocol (Polyplus Transfection,France). Concentration of siRNA for each experiment are indicated infigure legend. Scramble siRNA (Eurogentec, Belgium) was used as control.Cells were harvested 48 h later for RNA and extraction and RT-PCRanalysis.

Total RNA Extraction and cDNA Analysis

Total RNA was isolated using RNA easy kits (Qiagen, France) according tothe manufacturer's protocol. Cells were passed through a 22G syringeseveral times for disruption in the lysis buffer. Total RNA (1 μg) wassubmitted to reverse transcription using the Superscript III reversetranscriptase kit (Life Technologies) using hexamer primers. cDNA wereamplified by PCR under the following conditions: 96° C. for 5 min,cycles of 30 s at 96° C., 30 s at the appropriate temperature (58 to 62°C.), 30 s to 1 min at 72° C., and a final step of 7 min at 72° C. Twentyseven cycles were performed to amplify HPRT, 27 for semi-quantitativeanalysis of DNM2, 30 for restriction enzyme digestion of the DNM2amplicon. Sequences of all the primers used are indicated in thefollowing table:

Table: Primers used in this study Target Sequence (5′-3′) ApplicationhHPRT F ACCCCACGAAGTGTTGGATA Semi-quantitative analysis in human cells RAAGCAGATGGCCACAGAACT hDNM2 F GAAAAAGCAGGTCGTCAAGCSemi-quantitative analysis in human cells and R ATTGGGGATGGCTCTCTTPfoI Semi digestion profile Legend: F: Forward. R: Reverse. DNM2:Dynamin 2. HPRT: hypoxanthine guanine phosphoribosyl transferase

The half of the PCR products was used for restriction enzyme digestionwith 2U of BglI (New England Biolabs, France) for 2 h at 37° C. Imageacquisition of PCR products after agarose gel electrophoresis wasperformed using G-box (Ozyme, France) and associated-signal wasquantified using ImageJ Software (NIH; http://rsbweb.nih.gov/ij).

Results

Identification of Allele-Specific siRNA by Targeting a HeterozygousNon-Pathogenic Polymorphism

The patient-derived cells harbour the p.R465W DNM2 mutation and areheterozygous for the single nucleotide polymorphism (SNP) rs2229920 (Cand T) corresponding to one basis of the codon encoding the amino-acidat position 713 in the DNM2 protein sequence. By sequencing the DNM2mRNA, we showed that the mutation is in frame with the C version of thepolymorphism. The presence of the HTZ SNP allowed to screen forefficient AS-siRNA against the two possible versions of the SNP (C andT). Two siRNA against the C (including the mismatch at position 16 or17, i.e. si16-713C and si17-713C, respectively) and one siRNA againstthe T (including the mismatch at position 17 called si17-713T) wereassessed. Cells were transfected with 30 nM of siRNA for 48 hours beforeRT-PCR analysis. The 3 tested siRNA are efficient to decrease theexpression of the DNM2 transcript when compared to cells transfectedwith Scramble siRNA or non-transfected cells (FIG. 9A). Quantificationof the DNM2 transcript relative to HPRT mRNA used as housekeeping gene(FIG. 9B) indicates that a mismatch at position 17 are more efficientcompared to position 16. Using a RT-PCR assay developed to discriminatethe allele C and the allele T after restriction enzyme digestion ofamplicon, we assessed allele-specific properties of these 3 siRNA at 30nM for 48 hours.

Scramble siRNA-transfected cells and non-transfected cells show a C(Mutant)/T (WT) ratio equal to 1 in agreement with similar expression ofboth WT and mutated alleles (FIG. 10). The ratio decreases when siRNAagainst the C are used but increase when siRNA against the T is used(FIG. 10B left panel). Allele-specificity of these 3 siRNA was confirmedby quantifying the mRNA level harbouring the T nucleotide relative toHPRT expression showing a decrease only when the si17-713T is used (FIG.10B, right panel).

Modification of the Sequence of the Allele-Specific siRNA Against the CAllele of the Non-Pathogenic Polymorphism

With the objective to increase siRNA efficiency, length of the si17-713Cwas modified by adding nucleotides in 5′ or 3′ (FIG. 11A). Whentransfected at 50 nM for 48 hours, all the 3 modified siRNA are able todecrease expression of the DNM2 transcript compared to scramble siRNA(FIG. 11C) but, among them, the si713C-17/21 appears less efficient(although still of interest with around 30% of decrease).Allele-specificity was assessed using RT-PCR followed by BglI digestion.In non-transfected cells and in cells transfected with scramble siRNA,the calculated allele C/allele T ratio is around 1 in agreement withsimilar expression level of both alleles. As expected, transfection of50 nM of all the modified AS-siRNA against the C for 48 hours leads tothe decrease in the allele C/allele T ratio.

Discussion

In case of dominant diseases in which several causing mutations havebeen identified, two Allele-specific silencing by RNAi strategies may bedeveloped. The first strategy corresponds to personalized therapy bydeveloping AS-siRNA against each identified mutation as developed forthe p.R465W DNM2 mutation causing AD-CNM. The second strategy is todevelop AS-siRNA against a non-pathogenic variant associated with thedisease. Here, we show that this pan-mutation strategy may be developedfor the DNM2-linked CNM by targeting a common non-pathogenic singlenucleotide polymorphism (SNP) present in the coding region of the DNM2transcript. Given that this SNP is frequently found in heterozygousstate (C on one allele and T on the other one), AS-siRNA against the SNPenable silencing of all the mutated transcript by using the AS-siRNAagainst the version of the SNP in frame with the mutation.

Here, we have identified several efficient AS-siRNA against the twopossible version of the SNP rs2229920 (either the C allele or the Tallele). This results highlight for the first time the possibility totarget all the dominant DNM2 mutations whatever the resulting disease(CNM, CMT or HSP) and also to decrease, in a controlled manner, the DNM2expression in diseases associated with an overexpression of this protein(X-linked myotubular myopathy, pancreas and prostate cancers).

The invention claimed is:
 1. An allele specific siRNA (AS-siRNA) thatcomprises a sequence mismatch and is able to silence the expression ofonly one allele of a heterozygous DNM2 gene in a cell, wherein the DNM2gene is heterozygous for the presence of a non-pathologicalpolymorphism, wherein the non-pathological polymorphism is rs2229920 (Cor T) or rs12461992 (A or T), and wherein the AS-siRNA targets a regionof the allele comprising said non-pathological polymorphism.
 2. TheAS-siRNA of claim 1, wherein the AS-siRNA is of 19-23 base pairs inlength.
 3. The AS-siRNA of claim 1 wherein the DNM2 gene is heterozygousfor the presence of a disease-causing mutation.
 4. The AS-siRNA of claim3, wherein said non-pathological polymorphism is on the same allele asthe disease-causing mutation.
 5. The AS-siRNA of claim 3, wherein thedisease-causing mutation is 1393C>T; c.1105C>T, c.1106G>A, c.1856C>T orc.1948G>A.
 6. The AS-siRNA of claim 1, wherein the AS-siRNA is of 19base pairs in length and wherein the position of the sequence mismatchis located at position N16 or N17 from the 5′ end of the sense strand ofsaid AS-siRNA.
 7. The AS-siRNA of claim 1, wherein the AS-siRNAcomprises a sense strand selected from the group consisting of : SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
 8. The AS-siRNA of claim1, wherein the AS-siRNA reduces expression of DNM2 mRNA and/or DNM2protein by 20-60%.
 9. A vector encoding the AS-siRNA according toclaim
 1. 10. An isolated target cell, which is transfected or transducedwith the vector according to claim
 9. 11. An in vitro method forsilencing the expression of a mutated allele of a DNM2 gene withoutsilencing the expression of the wild type allele of the DNM2 gene in atarget cell, comprising introducing in said target cell an AS-siRNAaccording to claim 1 or a vector encoding the AS-siRNA.
 12. A method forameliorating a disease selected from autosomal dominant centronuclearmyopathy, myotubular myopathy, and Duchenne muscular dystrophy, in asubject in need thereof, comprising administering to the subject atherapeutic amount of the AS-siRNA of claim 1, or a vector encoding theAS-siRNA, or a cell transfected or transduced with the vector.
 13. AnAS-siRNA that comprises a sense strand selected from the groupconsisting of : SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQID NO:9 and SEQ ID NO:10.